The increasing prevalence of antibiotic resistance is compromising our ability to treat infection. Therefore, this application's broad, long-term objective is to develop and establish proof of principle for a new class of therapeutics targeting pathogen host interaction. Many bacterial pathogens use specialized secretion systems to inject virulence factors into host cells. These systems are absolutely required for virulence and therefore offer a promising therapeutic target. The aim of this proposal is to establish type IV secretion systems (T4SS) -- one of the major classes of such injection apparatuses - as a target for antimicrobial therapy. We recently completed a high throughput screen of a 235,000 compound library using the model pathogen, Legionella pneumophila, in order to identify small molecule inhibitors of T4SS. Here, we propose to follow up on preliminary data obtained from this screening effort with three specific aims. In the first, experiments are proposed to characterize previously identified, strong screening hits in order to confirm specificity, define potency, and characterize activity spectrum in L. pneumophila, Coxiella burnetii and a series of more distantly related T4SS-dependent pathogens. In the second, iterative structure-activity relationship studies will be used to identify analogues with the enhanced pharmacological potential. In the third, the activity of select small molecule inhibitors will be characterized in ivo in murine models of infection with L. pneumophila, C. burnetii, and Bartonella. Taken together these experiments should establish the theoretical basis for T4SS therapy and its potential use as a new human therapeutic.